PROJECT SUMMARY/ABSTRACT Cells must replicate their DNA with great fidelity during the cell cycle to ensure the production of healthy descendants. In human cells, replication begins at tens of thousands of sites known as replication origins. These origins fire at different times during the S-phase of the cell cycle. Furthermore, the pattern for firing of origins can vary for different cell types in the body as well as in cancer cells. Our overall goal is to understand how cells orchestrate the firing of replication origins to guarantee the orderly duplication of the DNA. This issue is important because derangements in origin firing can lead to genomic instability. Key steps in the initiation of replication involve binding of the minichromosome maintenance (MCM) complex to chromatin and subsequent activation of this complex to generate the active replicative helicase. This activation requires binding of the Cdc45 and GINS proteins to the MCM complex. In vertebrates, a protein known as Treslin and its partner MTBP act as chaperones that promote integration of Cdc45 and GINS with the MCM complex. This process depends on phosphorylation of Treslin by the S-phase cyclin-dependent kinase (S-Cdk), a key positive regulator of S-phase. Moreover, Treslin is also the target of a counteracting inhibitory mechanism that involves phosphorylation by the checkpoint-regulatory kinase Chk1. Thus, cells strictly regulate the ability of Treslin- MTBP to promote initiation. Our research aims to uncover novel aspects of how, where, and when cells initiate replication. First, we will investigate the roles of newly identified proteins that associate with the Treslin-MTBP complex in the regulation of initiation. These experiments may shed light on how the status of chromatin influences replication. Second, we will determine the binding sites for Treslin-MTBP throughout the genome and examine whether this binding helps to determine sites of initiation. These studies will test the hypothesis that a newly identified DNA-binding domain in MTBP promotes the targeting of Treslin-MTBP to origins. Finally, we will probe the mechanism by which Chk1 inhibits the Treslin-MTBP complex. The goal of these experiments would be to unravel how cells suppress initiation at certain locations and times to choreograph replication. These experiments will be carried out with both human tissue culture cells and Xenopus egg extracts. This strategy will take advantage of the complementary benefits of each system. These studies have the potential to generate valuable insights into how cells maintain the integrity of their genomes throughout life. This knowledge would be especially pertinent to human health. Disruption of genomic integrity as a result of defective replication as well as other perturbations can lead to numerous human pathologies, most notably cancer. Therefore, a comprehensive understanding of how cells replicate their DNA correctly will aid in the discovery of treatments for cancer and other diseases.